3 Materials and Methods
4.1 Assessment of mutant PrP C topology
4.1.1 Mutants in the hydrophobic core region of PrP C
During cellular trafficking and protein maturation, subpopulations of PrP insert into the membrane of the endoplasmic reticulum via the transmembrane domain (TM). In order to assess the function of the hydrophobic core (HC) within this domain on transmembrane topology, a set of mutants, bearing micro-deletions within the HC region, between codons 114 to 121 of the murine prion protein, had previously been constructed by Mr. Hartmut Niemann in the group. The deletion size and position varies between the different mutants, thereby enabling the assignment of possible functional effects to distinct amino acids within this region (Figure 17).
Figure 17: Mouse PrP deletion mutants
Schematic drawing of mouse PrPC. Below the scheme, the amino acid sequence displays the deletions within the hydrophobic core region for each mutant.
1-3: -helical structure; 1-2: -sheet structure; Cu: Copper binding sites; GPI-anchor signal sequence; Octa-R: octa-repeats; SS: signal sequence; TM: transmembrane domain
68 4.1.2 Topology assay
In order to assess the effect of these mutations at the ER membrane, the various deletion mutants were translated in vitro in the presence of ER-derived microsomal membranes, followed by the addition of proteinase K (PK). Whereas any parts of the protein residing in the lumen of the endoplasmic reticulum are protected by the ER membrane against PK-mediated degradation, the digestion of the cytosolic protein parts by PK leads to the truncation of the transmembrane forms of PrPC, either at the N-terminus (for CtmPrP) or at the C-terminus (for
NtmPrP), resulting in a conformation (Hegde et al., 1998a) (Figure 18).
Figure 18: Scheme of the topology assay
(a) Principle of the in vitro translation assay: Starting with a plasmid template, DNA is transcribed and translated in vitro. In the presence of microsomal membranes, the polypeptide can be translocated in the ER lumen, resembling the situation at the ER in live cells. Modified from: (Ambion, 2008)
(b) Truncation of fragments: After translation, proteinase K (PK) is applied to the reaction, digesting all cytosolic parts of the proteins. Only the luminal parts are protected from PK digestion, resulting in distinct sizes for the different topological PrP forms. Modified from: (Hegde et al., 1998a)
(c) Final analysis: After further purification steps (i.e., IP or isolation of microsomal membranes by ultracentrifugation) the different peptide fragments can be analyzed by SDS-PAGE and autoradiography.
As an example, the ratios of the different topological forms of human PrP-wt and PrP-A117V are shown.
From: (Hegde et al., 1998a)
Results
69 4.1.3 Characterization of PrPC mutant topology
The PrP fragments, generated in the topology assay were characterized by subsequent immunoprecipitation with different antibodies, directed against the N- or C-terminus of PrPC. These antibodies selectively discriminated the different topological PrP forms due to the loss of their epitope for the N-terminal (in the case of CtmPrP) or the C-terminal (for NtmPrP) antibody after digestion by proteinase K (Figure 19b).
As expected, an antibody directed against the N-terminus of PrPC precipitated full-length
secPrP (secretory PrP) and the transmembrane fragment NtmPrP. Fragments that comprise an intact C-terminal region, such as secPrP and CtmPrP, on the other hand, were immunoprecipitated with the C-terminal antibody 6H4 (Figure 19a). In addition to these expected fragments, immunoprecipitation with the C-terminal antibody revealed an as yet uncharacterized fragment of 17-18 kDa. This N-terminally truncated fragment already appeared in the absence of PK (Figure 19a, lane 1) and is therefore not a truncated transmembrane form generated by PK digestion. Size and truncation of the N-terminus rather correspond to a fragment, which is created by -cleavage (aa 109-111) during the normal metabolism of PrPC, termed C1 (Chen et al., 1995).
In addition, in vitro translated hamster PrP-wt, harboring the epitope for the antibody 3F4 (aa 109-112) was immunoprecipitated using this antibody (data not shown). In contrast to the other topological forms, 3F4 was unable to immunoprecipitate this N-terminal truncated fragment. This observation strengthens the conclusion, that the N-terminal truncated fragment is indeed C1, since PrPC -cleavage takes place at the same site and leads therefore to the destruction of the 3F4 epitope.
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Figure 19: Characterization of PrPC fragments
(a) After in vitro translation, samples were treated with PK as indicated, purified by either ultracentrifugation (UC) or by immunoprecipitation (IP), separated by SDS-PAGE and visualized by autoradiography.
Lane 1: Positive control: The C1 fragment appears already without PK treatment.
Lane 2: Detergent control: Application of detergent during PK-digestion leads to the loss of microsomal membrane integrity and thus to the loss of protection against PK.
Lanes 3 and 6: PrP-wt or PrP 114-121, UC-purified. In contrast to PrP-wt, PrP 114-121 displays massive reduction of the two TM-fragments and the C1-fragment.
Lanes 4 and 7: IP with N-Ab. Only fragments with an intact N-terminus remain detectable (secPrP and
NtmPrP).
Lanes 5 and 8: IP with C-Ab. Only fragments with an intact C-terminus remain detectable (secPrP, CtmPrP and C1).
(b) Scheme for the characterization of PrPC fragments by IP, using antibodies for different protein regions.
Due to the truncations after PK-digestion or -cleavage, an antibody that recognizes an epitope in the N-terminus of PrPC (N-Ab) can no longer bind to fragments that have been digested at the N-terminus such as CtmPrP or C1. Vice versa, C-terminally truncated fragments, such as NtmPrP, cannot be immunoprecipitated with a C-terminally binding antibody (C-Ab)
Abbreviations: AP: Acceptor peptide (AP), preventing protein glycosylation; C-Ab: C-terminal binding antibody; Det: Application of detergent in the translation assay, which disrupts the integrity of the microsomal membranes; IP: Immunoprecipitation; Mem: Microsomal membranes; N-Ab: N-terminal binding antibody;
Pur: Method of peptide purification; UC: Ultracentrifugation
Results
71 Differences in the metabolism of PrPC fragments
In order to further characterize the identity of the different fragments, PrP wild-type and mutant constructs were translated for different time periods. For the wild-type construct, the amount of the N-terminally truncated fragment increased over time, whereas the proportion of the transmembrane fragments remained unchanged (Figure 20). Since the rate of in vitro protein synthesis slows down over time due to the depletion of essential components, an increase in the amount of a distinct fragment is unlikely to result from elevated protein synthesis, but is rather due to protein degradation, such as proteolytic cleavage. This observation therefore strengthens the view that the 17-18 kDa fragment is indeed C1, produced by -cleavage.
In comparison to PrP-wt, the deletion mutant PrP 114-121 displayed almost complete loss of the transmembrane PrP topologies CtmPrP and NtmPrP, as well as of the C1 fragment (Figure 19a; Figure 20a, b).
Figure 20: Time course of C1 accumulation
(a) Prnp-wt and Prnp Δ114-121 were translated for 45, 90 and 180 minutes. Thereafter the samples were digested with PK and purified by ultracentrifugation.
(b) Quantification of (a). Over time, only the amount of C1 increases in PrP-wt whereas the proportion of the transmembrane topology remained unchanged.
72 4.1.5 Impact of the hydrophobic core on PrPC topology and -cleavage In order to study the influence of the hydrophobic core (HC) on PrPC membrane topology and
-cleavage in more detail, the various deletion mutants were analyzed in the topology assay.
Except for PrP 116-119, all deletion mutants showed a significant decrease in NtmPrP and
CtmPrP forms compared to PrP-wt (Figure 21, Table 9). In addition to the decrease of transmembrane topology, deletions in the core region also markedly affected PrP -cleavage as demonstrated by the decrease in C1 levels.
Figure 21: Topology assessment of the different PrPC deletion mutants
(a) Fragments generated by the topology assay, purified by UC and separated by SDS-PAGE.
Lanes 2-10: Topology assay with PrP-wt and the different mutants. The deletions within HC caused a significant decrease in TM-topology compared to PrP-wt. In addition, these deletions also had an impact on the -cleavage of PrPC resulting in changes in C1-fragment expression levels. Lanes 1, 11: Controls.
(b) Quantification of PrPC topology. Signal intensities of the different PrP fragments were quantified by densitometry. For each mutant, the mean value of at least three independent experiments is shown.
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wt Δ114-115 Δ114-117 Δ114-119 Δ114-121 Δ116-119 Δ116-121 Δ118-121 Δ120-121
Ctm
*** *** *** *** * *** *** ***
Ntm
** ** *** *** n.s. *** *** ***
Sec
*** *** *** *** n.s. *** ** n.s.
C1
** *** *** *** n.s. *** n.s. n.s.
Table 9: Statistical analysis of mutant PrP topology
The statistical significance of the differences in fragment expression for each mutant compared to PrPC-wt was calculated using one-way ANOVA in combination with Tukey’s multiple comparison test (* denotes p<0.05; **
denotes p<0.01; *** denotes p<0.001; n.s. = not significant)
74 4.1.6 Correlations of PrPC topology with HC sequence and hydrophobicity With regard to the differences of PrPC topology and -cleavage between the different deletion mutants, the number of deleted amino acids alone did not account for the changes in PrP transmembrane topology. Deleting amino acids 116-119, for example, did not lead to a reduction of CtmPrP and caused only a slight decrease in NtmPrP. On the other hand, a deletion of the same size at the positions 114-117 or 118-121 resulted in a markedly reduced quantity of transmembrane PrP forms (Table 10). Thus, the strong differences in the quantity of transmembrane PrP molecules between the various mutants were not primarily determined by the deletion size or the amino acid sequence within the HC. The efficiency of the different deletion mutants to be incorporated in the ER membrane rather correlated with the altered hydrophobicity of the deletion region between amino acids 114-121 (Figure 22a), indicating that the main impact of HC on PrP membrane topology is based on its hydrophobic character.
By contrast, -cleavage of PrP did not show a significant correlation with the hydrophobic character of HC but was rather determined by its amino acid sequence (Figure 22b). In the mutant PrP 120-121, for example, -cleavage efficiency was completely maintained;
indicating that only the segment 114-119 is implicated in PrPC -cleavage (Table 10). This hypothesis was substantiated by the significant correlation between the sequence similarity of the amino acids 119 and the amount of C1 (Figure 22b). The segments 120 or 114-121 exhibited a much weaker correlation between the similarity and -cleavage (data not shown), further implying that only the amino acids of the palindromic sequence (aa 114-119) are necessary for PrP -cleavage.
Results
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Table 10: Characteristics of PrPC mutants
For each PrPC mutant, the amino acid sequence within the region of deletion from position 114-121 is shown.
Amino acids in bold letters are identical to the PrP-wt sequence.
Each amino acid within this region was characterized by its hydrophobic or hydrophilic tendency (=
hydropathy). The overall hydrophobic score of the region is given as the sum of the hydropathy values for the remaining non-deleted amino acids of codons 114-121. Furthermore, the similarity of PrP-wt and mutant sequences of the amino acids 114-119 is shown next to the proportion of the transmembrane versions (CtmPrP plus NtmPrP) and of the C1 fragment.
Figure 22: Correlation of mutant topology and -cleavage with HC sequence and hydrophobicity (a) The hydrophobicity of the amino acids of the HC-region 114-121, assessed by their hydropathy index
(abscissa) correlated with the amount of transmembrane PrPC topology (left) but not with the amount of PrP -cleavage (right).
(b) In contrast to the changes in transmembrane topology (left), only the cleavage of the C1-fragment (right) correlated with the similarity (abscissa) of the HC wt-sequence.
TM Hydropathy Similarity
114 115 116 117 118 119 120 121 114-121 114-119
wt A A A A G A V V 17.0 49.0 15.5 ± 1.6 36.6 ± 6.7
114 2 A A G A V V G G 13.4 13.0 6.1 ± 1.6 26.1± 5.0
114 4 G A V V G G L G 9.8 11.0 4.0 ± 1.6 10.9 ± 4.6
114 6 V V G G L G G Y 8.4 -10.0 3.7 ± 0.6 4.1 ± 0.6
114 8 G G L G G Y M L 0.0 -3.0 2.8 ± 1.7 1.5 ± 0.9
116 4 A A V V G G L G 12.0 20.0 11.3 ± 1.6 36.4 ± 3.4
116 6 A A G G L G G Y 3.6 5.0 0.9 ± 0.9 12.0 ± 5.5
118 4 A A A A G G L G 7.2 40.0 4.2 ± 0.6 33.1 ± 4.8
120 2 A A A A G A G G 8.6 49.0 4.1 ± 1.8 38.4 ± 4.5
Amino acid position
TM fragments (%) C1 fragment (%)
76 4.1.7 Impact of PrPC topology and -cleavage on prion disease
Two hereditary forms of human prion diseases are associated with missense mutations in the hydrophobic core of PrP, i.e., G114V and A117V (Rodriguez et al., 2005). It has already been shown that the mutation A117V exhibited an increase in transmembrane PrP topology, especially CtmPrP (Hegde et al., 1998a). These findings suggest that the pathologic impact of mutations within TM could be associated with alterations in the metabolism of PrPC, such as the upregulation of a toxic transmembrane PrP form, i.e., CtmPrP.
The in vitro topology assay also revealed for G113V (corresponding to the pathologic human mutation G114V) a significant threefold increase of CtmPrP topology from 10.5% to 32.7%, whereas PrPC -cleavage was significantly reduced to 50% of the wild-type level
Figure 23), indicating that alterations in -cleavage as well as in CtmPrP formation might contribute to the pathology of prion diseases.
Figure 23: Assessment of pathologic G113V metabolism
(a) Fragments generated by the topology assay, purified by UC and separated by SDS-PAGE.
(b) Quantification of PrPC topology. Signal intensities of the different PrP fragments were quantified by densitometry. For each mutant, the mean value of at least three independent experiments is shown. The mutant G113V expressed half the amount of C1 fragment compared to PrP-wt (p<0.001), whereas CtmPrP was three-fold increased (p<0.001).
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